Mesenchymal stem cells for cartilage repair

ABSTRACT

It has been discovered that mesenchymal stem cells (MSCs) in a polymeric carrier implanted into a cartilage and/or bone defect will differentiate to form cartilage and/or bone, as appropriate. Suitable polymeric carriers include porous meshes or sponges formed of synthetic or natural polymers, as well as polymer solutions. A presently preferred material is a polyglycolic acid mesh.

BACKGROUND OF THE INVENTION

The present invention is generally in the area of regeneration andrepair of cartilage, and more particularly relates to implantation ofmesenchymal stem cells on matrices to form cartilage.

Arthritis, both rheumatoid and osteoarthritis, constitutes a majormedical problem. In particular, degeneration of articular cartilage inosteoarthritis is a serious medical problem. Drugs are given to controlthe pain and to keep the swelling down, but the cartilage continues tobe destroyed. Eventually, the joint must be replaced. As reviewed byMankin, N.E. J. Med. 331(14), 940-941 (October 1994), it is stillunknown why cartilage does not heal and no solutions to this problem areknown.

Whether articular cartilage is damaged from trauma or congenitalanomalies, its successful clinical regeneration is poor at best, asreviewed by Howell, et al. Osteoarthritis: Diagnosis and Management, 2nded., (Philadelphia, W. B. Saunders, 1990) and Kelley, et al. Textbook ofRheumatology, 3rd ed., (Philadelphia, W. B. Saunders, 1989) 1480. Theinability of adult articular cartilage for self repair has been wellrecognized and has stimulated major interest. There are two majormechanisms of articular cartilage repair: intrinsic and extrinsic,discussed by Edwards Proc. Ins. Mech. Eng. 181, 16 (1967), and SokoloffJ. Rheumatol. 1, 1 (1974).

Superficial or partial-thickness injuries that do not penetrate thesubchondral bone rely on the intrinsic mechanism for repair. Soon aftersuperficial injury, chondrocytes adjacent to the injured surfaces show abrief burst of mitotic activity associated with an increase inglycosaminoglycan and collagen synthesis. Despite these attempts atrepair, there is no appreciable increase in the bulk of cartilage matrixand the repair process is rarely effective in healing the defects.

Osteochondral, or full-thickness, cartilage defects extend into thesubchondral bone. Such defects arise after the detachment ofosteochondritic dissecting flaps, fractured osteochondral fragments, orfrom chronic wear of degenerative articular cartilage. Osteochondraldefects depend on the extrinsic mechanism for repair. Extrinsic healingrelies on mesenchymal elements from subchondral bone to participate inthe formation of new connective tissue. This fibrous tissue may or maynot undergo metaplastic changes to form fibrocartilage. Even iffibrocartilage is formed, it does not display the same biochemicalcomposition or mechanical properties of normal articular cartilage orsubchondral bone and degenerates with use, Furukawa, et al., J. BoneJoint Surg. 62A, 79 (1980); Coletti, et al., J. Bone Joint Surg. 54A,147 (1972); Buckwalter, et al., "Articular cartilage: composition,structure, response to injury and methods of facilitating repair", inArticular Cartilage and Knee Joint Function: Basic Science andArthroscopy, Ewing J E, Ed., (New York, Raven Press, 1990), 19. Theensuing osteoarthritis may result in permanent disability and discomfortto the patient.

As described in U.S. Pat. No. 5,041,138 to Vacanti, et al., and U.S.Pat. No. 4,846,835 to Grande, cartilage has been grown by seedingsynthetic polymeric matrices with dissociated cells, which are thenimplanted to form new cartilage. Cartilage has also been grown from aninjected or implanted ionically crosslinked hydrogel-chondrocytesuspension, as described by Atala, et al., J. Urology vol. 150, no. 2,part 2, 745-747 (August 1993). Injection of dissociated chondrocytesdirectly into a defect has also recently been described as a means forforming new cartilage, as reported by Brittberg, et al., N. E. J. Med.331, 889-895 (October 1994). Cartilage was harvested from minorload-bearing regions on the upper medial femoral condyle of the damagedknee, cultured, and implanted two to three weeks after harvesting.

Freed and Grande, J. Biomed. Mater. Res. 28, 891 (1994) cultured maturechondrocytes from New Zealand white rabbits in vitro onto polyglycolicacid (PGA) scaffolds for 21/2 weeks. A full thickness articularcartilage defect was then created in the femoropatellar groovebilaterally in syngeneic New Zealand white rabbits. Mature chondrocyteson the PGA-matrix (PGA-cells) were imbedded into one knee joint whilePGA discs alone were imbedded into the contralateral knee and theanimals euthanized at one and six months post-implantation. The repairtissue was well bonded to the host tissue and the surfaces of thesedefects were congruent with the host cartilage. The PGA alone showed amixture of fibrocartilage and hyaline cartilage oriented randomly. ThePGA-cells implant showed normal articular cartilage histology, but didnot have normal subchondral bone.

A disadvantage of these systems is that the chondrocytes must beobtained from the patient, typically by a biopsy, cultured, and thenimplanted on the matrix. This is relatively easy in laboratory animals,but presents greater logistical problems in humans where a defect iscreated by the biopsy required to provide cells for repair of anotherdefect. Moreover, if the defect includes a part of the underlying bone,this is not corrected using chondrocytes, which are alreadydifferentiated and will not form new bone. The bone is required tosupport the new cartilage.

Stem cells are defined as cells which are not terminally differentiated,which can divide without limit, and divides to yield cells that areeither stem cells or which irreversibly differentiate to yield a newtype of cell. Those stem cells which give rise to a single type of cellare call unipotent cells; those which give rise to many cell types arecalled pluripotent cells. Chondro/osteoprogenitor cells, which arebipotent with the ability to differentiate into cartilage or bone, wereisolated from bone marrow (for example, as described by Owen, J. CellSci. Suppl. 10, 63-76 (1988) and in U.S. Pat. No. 5,226,914 to Caplan,et al.). These cells led Owen to postulate the existence of pluripotentmesenchymal stem cells, which were subsequently isolated from muscle(Pate, et al., Proc. 49th Ann. Sess. Forum Fundamental Surg. Problems587-589 (Oct. 10-15, 1993)), heart (Dalton, et al., J. Cell Biol. 119,R202 (March 1993)), and granulation tissue (Lucas, et al., J. CellBiochem. 122, R212 (March 1993)). Pluripotency is demonstrated using anon-specific inducer, dexamethasone (DMSO), which elicitsdifferentiation of the stem cells into chondrocytes (cartilage),osteoblasts (bone), myotubes (muscle), adipocytes (fat), and connectivetissue cells.

Unfortunately, although it is highly desirable to have stem cells whichare easily obtained by a muscle biopsy, cultured to yield large numbers,and can be used as a source of chondrocytes or osteoblasts or myocytes,there is no known specific inducer of the mesenchymal stem cells thatyields only cartilage. In vitro studies in which differentiation isachieved yields a mixture of cell types. Studies described in U.S. Pat.Nos. 5,226,914 and 5,197,985 to Caplan, et al., in which the cells wereabsorbed into porous ceramic blocks and implanted yielded primarilybone. Studies using bone morphogenic protein-2 (rhBMP-2) in vivo alwaysyield an endochondral bone cascade. That is, cartilage is formed first,but this cartilage hypertrophies, is invaded by vasculature andosteoblasts, and is eventually replaced by bone complete with marrow(Wozney, Progress in Growth Factor Research 1, 267-280 (1989)). Studiestesting rhBMP-2 on the mesenchymal stem cells in vitro produced mixturesof differentiated cells, although cartilage predominated (Dalton, etal., J. Cell Biol. 278, PZ202 (February 1994)). Incubation ofmesenchymal cell cultures with insulin led to a mixed myogenic andadipogenic response, while incubation with insulin-like growth factors Ior II led to a primarily myogenic response (Young, et al., J. CellBiochem. 136, CD307 (April 1992)). U.S. Pat. Nos. 4,774,322 and4,434,094 to Seyedin, et al., report the isolation of a factor thatinduces an osteogenic response in vivo or cartilage formation in vitrowhen mixed with muscle cells.

It is therefore an object of the present invention to provide a methodand compositions for formation of cartilage.

It is a further object of the present invention to provide a method andcompositions to induce differentiation of mesenchymal stem cells intocartilage.

SUMMARY OF THE INVENTION

It has been discovered that mesenchymal stem cells (MSCs) in a polymericcarrier implanted into a cartilage and/or bone defect will differentiateto form cartilage and/or bone, as appropriate.

Suitable polymeric carriers include porous meshes or sponges formed ofsynthetic or natural polymers, as well as polymer solutions. A presentlypreferred material is a polyglycolic acid mesh.

As demonstrated by the examples, MSCs were isolated from adult rabbitmuscle and cultured in vitro in porous polyglycolic acid polymermatrices. The matrices were implanted into three mm diameter fullthickness defects in rabbit knees with empty polymer matrices serving asthe contralateral controls. The implants were harvested six and 12 weekspost-op. At six weeks, the controls contained fibrocartilage while theexperimentals contained undifferentiated cells. By 12 weeks post-op, thecontrols contained limited fibrocartilage and extensive connectivetissue, but no subchondral bone. In contrast, the implants containingMSCs had a surface layer of cartilage approximately the same thicknessas normal articular cartilage and normal-appearing subchondral bone.There was good integration of the implant with the surrounding tissue.Implantation of MSCs into cartilage defects effected repair of both thearticular cartilage and subchondral bone.

DETAILED DESCRIPTION OF THE INVENTION I. Isolation and Preparation ofMSCs

Mesenchymal stem cells (MSCs) are isolated from connective tissue,including muscle and dermis. They have advantages based on theirunlimited growth potential and their ability to differentiate intoseveral phenotypes of the mesodermal lineage, including cartilage andbone.

MSCs are preferably isolated from muscle using a standard punch ordermal biopsy. However, MSCs can be obtained from bone marrow or othermesenchymal tissues.

A detailed procedure for isolation of MSCs from embryonic chick muscleis described by Young, et al., J. Tiss. Cult. Meth. 14, 85-92 (1992),the teachings of which are incorporated by reference herein. The samebasic procedure is used for isolation of mammalian MSCs from muscle.Muscle is removed, rinsed, minced and the cells isolated by digestionwith collagenase/dispase and cultured in gelatin-coated dishes in EMEMor DMEM media with pre-selected horse serum (serum is pre-screened forsupport of MSCs but not fibroblasts) until confluent. The cells aretrypsinized and slowly frozen in freezing chambers 7.5% DMSO at -80° C.The cells are then thawed and cultured in the same media without DMSO.Freezing is used to kill any fibroblasts present in the cell culture.Filtration through 20 micron Nitex is used to remove myotubes. Reagentscan be obtained from Sigma Chemical Co., St. Louis, Mo. or GIBCO, GrandIsland, N.Y.

II. Polymeric Matrices

There are basically two types of matrices that can be used to supportthe MSCs as they differentiate into cartilage or bone. One form ofmatrix is a polymeric mesh or sponge; the other is a polymeric hydrogel.In the preferred embodiment, the matrix is biodegradable over a timeperiod of less than a year, more preferably less than six months, mostpreferably over two to ten weeks. In the case of joint surfaceapplication, the degradation period is typically about twelve totwenty-four weeks. In the case where weight bearing or high shear stressis not an issue, the degradation period is typically about five to tenweeks. The term bioerodible or biodegradable, as used herein, means apolymer that dissolves or degrades within a period that is acceptable inthe desired application, less than about six months and most preferablyless than about twelve weeks, once exposed to a physiological solutionof pH 6-8 having a temperature of between about 25° C. and 38° C. Thepolymer composition, as well as method of manufacture, can be used todetermine the rate of degradation. For example, mixing increasingamounts of polylactic acid with polyglycolic acid decreases thedegradation time.

A. Fibrous Matrices

Polymeric Materials

Fibrous matrices can be manufactured or constructed using commerciallyavailable materials. The matrices are typically formed of a natural or asynthetic polymer. Biodegradable polymers are preferred, so that thenewly formed cartilage can maintain itself and function normally underthe load-bearing present at synovial joints. Polymers that degradewithin one to twenty-four weeks are preferable. Synthetic polymers arepreferred because their degradation rate can be more accuratelydetermined and they have more lot to lot consistency and lessimmunogenicity than natural polymers. Natural polymers that can be usedinclude proteins such as collagen, albumin, and fibrin; andpolysaccharides such as alginate and polymers of hyaluronic acid.Synthetic polymers include both biodegradable and non-biodegradablepolymers. Examples of biodegradable polymers include polymers of hydroxyacids such as polylactic acid (PLA), polyglycolic acid (PGA), andpolylactic acid-glycolic acid (PLGA), polyorthoesters, polyanhydrides,polyphosphazenes, and combinations thereof. Non-biodegradable polymersinclude polyacrylates, polymethacrylates, ethylene vinyl acetate, andpolyvinyl alcohols. These should be avoided since their presence in thecartilage will inevitably lead to mechanical damage and breakdown of thecartilage.

Matrix Construction

In the preferred embodiment, the polymers form fibers which areintertwined, woven, or meshed to form a matrix having an interstitialspacing of between 100 and 300 microns. Meshes of polyglycolic acid thatcan be used can be obtained from surgical supply companies such asEthicon, N.J. Sponges can also be used. As used herein, the term"fibrous" refers to either a intertwined, woven or meshed matrix or asponge matrix.

The matrix is preferably shaped to fill the defect. In most cases thiscan be achieved by trimming the polymer fibers with scissors or a knife;alternatively, the matrix can be cast from a polymer solution formed byheating or dissolution in a volatile solvent.

Application of the Cells

The MSCs are seeded onto the matrix by application of a cell suspensionto the matrix. This can be accomplished by soaking the matrix in a cellculture container, or injection or other direct application of the cellsto the matrix. Media should be washed from the cells and matrix prior toimplantation.

The matrix seeded with cells is implanted at the site of the defectusing standard surgical techniques. The matrix can be seeded andcultured in vitro prior to implantation, seeded and immediatelyimplanted, or implanted and then seeded with cells. In the preferredembodiment, cells are seeded onto and into the matrix and cultured invitro for between approximately sixteen hours and two weeks. It is onlycritical that the cells be attached to the matrix. Two weeks is apreferred time for culture of the cells, although it can be longer. Celldensity at the time of seeding or implantation should be approximately25,000 cells/mm³.

B. Hydrogel Matrices

Hydrogel matrices are described, for example, in PCT US94/04710 byMassachusetts Institute of Technology and Childrens Medical CenterCorporation, the teachings of which are incorporated herein.

Polymers that can form ionic or covalently crosslinked hydrogels whichare malleable are used to encapsulate cells. For example, a hydrogel isproduced by cross-linking the anionic salt of polymer such as alginicacid, a carbohydrate polymer isolated from seaweed, with calciumcations, whose strength increases with either increasing concentrationsof calcium ions or alginate. The alginate solution is mixed with thecells to be implanted to form an alginate suspension. Then thesuspension is injected directly into a patient prior to hardening of thesuspension. The suspension then hardens over a short period of time dueto the presence in vivo of physiological concentrations of calcium ions.

The polymeric material which is mixed with cells for implantation intothe body should form a hydrogel. A hydrogel is defined as a substanceformed when an organic polymer (natural or synthetic) is cross-linkedvia covalent, ionic, or hydrogen bonds to create a three-dimensionalopen-lattice structure which entraps water molecules to form a gel.Examples of materials which can be used to form a hydrogel includepolysaccharides such as alginate, polyphosphazines, and polyacrylates,which are crosslinked ionically, or block copolymers such as Pluronics™or Tetronics™, polyethylene oxide-polypropylene glycol block copolymerswhich are crosslinked by temperature or pH, respectively. Othermaterials include proteins such as fibrin, polymers such aspolyvinylpyrrolidone, hyaluronic acid and collagen.

In general, these polymers are at least partially soluble in aqueoussolutions, such as water, buffered salt solutions, or aqueous alcoholsolutions, that have charged side groups, or a monovalent ionic saltthereof. Examples of polymers with acidic side groups that can bereacted with cations are poly(phosphazenes), poly(acrylic acids),poly(methacrylic acids), copolymers of acrylic acid and methacrylicacid, poly(vinyl acetate), and sulfonated polymers, such as sulfonatedpolystyrene. Copolymers having acidic side groups formed by reaction ofacrylic or methacrylic acid and vinyl ether monomers or polymers canalso be used. Examples of acidic groups are carboxylic acid groups,sulfonic acid groups, halogenated (preferably fluorinated) alcoholgroups, phenolic OH groups, and acidic OH groups.

Examples of polymers with basic side groups that can be reacted withanions are poly(vinyl amines), poly(vinyl pyridine), poly(vinylimidazole), and some imino substituted polyphosphazenes. The ammonium orquaternary salt of the polymers can also be formed from the backbonenitrogens or pendant imino groups. Examples of basic side groups areamino and imino groups.

Alginate can be ionically cross-linked with divalent cations, in water,at room temperature, to form a hydrogel matrix. Due to these mildconditions, alginate has been the most commonly used polymer forhybridoma cell encapsulation, as described, for example, in U.S. Pat.No. 4,352,883 to Lim. In the Lim process, an aqueous solution containingthe biological materials to be encapsulated is suspended in a solutionof a water soluble polymer, the suspension is formed into droplets whichare configured into discrete microcapsules by contact with multivalentcations, then the surface of the microcapsules is crosslinked withpolyamino acids to form a semipermeable membrane around the encapsulatedmaterials.

Polyphosphazenes are polymers with backbones consisting of nitrogen andphosphorous separated by alternating single and double bonds. Thepolyphosphazenes suitable for cross-linking have a majority of sidechain groups which are acidic and capable of forming salt bridges withdi- or trivalent cations. Examples of preferred acidic side groups arecarboxylic acid groups and sulfonic acid groups. Polymers can besynthesized that degrade by hydrolysis by incorporating monomers havingimidazole, amino acid ester, or glycerol side groups. For example, apolyanionic poly bis(carboxylatophenoxy)!phosphazene (PCPP) can besynthesized, which is cross-linked with dissolved multivalent cations inaqueous media at room temperature or below to form hydrogel matrices.

The water soluble polymer with charged side groups is ionicallycrosslinked by reacting the polymer with an aqueous solution containingmultivalent ions of the opposite charge, either multivalent cations ifthe polymer has acidic side groups or multivalent anions if the polymerhas basic side groups. The preferred cations for cross-linking of thepolymers with acidic side groups to form a hydrogel are divalent andtrivalent cations such as copper, calcium, aluminum, magnesium,strontium, barium, zinc, and tin, although di-, tri- or tetra-functionalorganic cations such as alkylammonium salts, e.g., R₃ N⁺ -\/\/\/-⁺ NR₃can also be used. Aqueous solutions of the salts of these cations areadded to the polymers to form soft, highly swollen hydrogels andmembranes. The higher the concentration of cation, or the higher thevalence, the greater the degree of cross-linking of the polymer.Concentrations from as low as 0.005 M have been demonstrated tocross-link the polymer. Higher concentrations are limited by thesolubility of the salt.

The preferred anions for cross-linking of the polymers to form ahydrogel are divalent and trivalent anions such as low molecular weightdicarboxylic acids, for example, terepthalic acid, sulfate ions andcarbonate ions. Aqueous solutions of the salts of these anions are addedto the polymers to form soft, highly swollen hydrogels and membranes, asdescribed with respect to cations.

Cell Suspensions

Preferably the polymer is dissolved in an aqueous solution, preferably a0.1 M potassium phosphate solution, at physiological pH, to aconcentration forming a polymeric hydrogel, for example, for alginate,of between 0.5 to 2% by weight, preferably 1%, alginate. The isolatedcells are suspended in the polymer solution to a concentration ofbetween 1 and 10 million cells/ml, most preferably between 10 and 20million cells/ml.

Methods of Implantation

In the preferred embodiment, the cells are mixed with the hydrogelsolution and injected directly into a site where it is desired toimplant the cells, prior to hardening of the hydrogel. However, thematrix may also be molded and implanted in one or more different areasof the body to suit a particular application. This application isparticularly relevant where a specific structural design is desired orwhere the area into which the cells are to be implanted lacks specificstructure or support to facilitate growth and proliferation of thecells.

The site, or sites, where cells are to be implanted is determined basedon individual need, as is the requisite number of cells. One could alsoapply an external mold to shape the injected solution. Additionally, bycontrolling the rate of polymerization, it is possible to mold thecell-hydrogel injected implant like one would mold clay.

Alternatively, the mixture can be injected into a mold, the hydrogelallowed to harden, then the material implanted.

The suspension can be injected via a syringe and needle directly into aspecific area wherever a bulking agent is desired, especially softtissue defects. The suspension can also be injected as a bulking agentfor hard tissue defects, such as bone or cartilage defects, eithercongenital or acquired disease states, or secondary to trauma, burns, orthe like. An example of this would be an injection into the areasurrounding the skull where a bony deformity exists secondary to trauma.The injection in these instances can be made directly into the neededarea with the use of a needle and syringe under local or generalanesthesia.

The suspension can be injected percutaneously by direct palpation.Alternatively, the suspension can be injected through a catheter orneedle with fluoroscopic, sonographic, computer tomography, magneticresonance imaging or other type of radiologic guidance.

III. Conditions to be Treated

The mesenchymal stem cells in and/or on a polymeric carrier can be usedto create or supplement connective tissue as required. In some cases,this will be to repair existing defects, for example, worn or torncartilage in joint linings. In other cases, it may be to create newtissue that performs a distinct function, such as to block tubes such asthe fallopian tubes or vas derens, or to decrease reflux due to urineleakage arising from incorrect placement of the ureter into the bladder.The selection of the form of the matrix, as well as the composition,will in many cases be determined by the function to be achieved, asdiscussed above.

Examples of situations in which new connective tissue is particularlydesirable, in addition to cartilage replacement or supplementation,include reconstruction of the spine, pubic symphysis ortemporomandibular joint (TMJ).

In some cases, it may be desirable to induce a mixed cell tissue, forexample, in breast reconstruction. Breast tissue is naturally composedof fat, cartilage and other connective tissue, muscle and other tissues.New breast tissue can be formed by implanting mesenchymal cells in apolymeric carrier in a fascial plane formed of muscle cells, fat,fibroblasts, and cartilage.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLE 1 Implantation of MSCs on PGA Scaffolds and Implantation intoFull Thickness Articular Cartilage Defects in Rabbits

This experiment was conducted to determine the regenerative capabilitiesof MSCs cultured on the PGA scaffolds and placed into syngeneic rabbitfull thickness articular cartilage defects.

Materials and Methods

New Zealand white rabbits were purchased from Hazelton (Denver, Pa.).Polyglycolic acid (PGA) discs, non-woven fiber mats, 1 cm diameter×0.2cm thick, composed of 12-14 μm diameter fibers at a density of 55-65mg/cm³ and sterilized with ethylene oxide were obtained from AlbanyInternational, Mansfield, Mass.

Rabbit MSCs were isolated as described by Pate et al Surgical Forum 44,587 (1993). Briefly, adult rabbit leg skeletal muscle was harvestedunder sterile conditions and placed in minimal essential medium withEarle's salts (EMEM) supplemented with 3×antibiotic-antimycotic solutionfor at least 10 minutes. The muscle was then finely minced withscissors. The media and tissue were centrifuged at 150×g for 10 minutes,the supernatant was discarded, and the tissue was transferred to asterile bottle containing a magnetic stir bar. The tissue was thendigested with a collagenase/dispase solution consisting of 250 U/mlWorthington CLSI collagenase, Freehold, N.J., and 33 U/ml CollaborativeResearch dispase, Cambridge, Mass., in the ratio of 1:4:15 (v/v/v) oftissue:collagenase/dispase:EMEM. Digestion required approximately 45minutes. The digested tissue was then centrifuged at 300×g for 20minutes, the supernatant discarded, and the cell pellet resuspended inEMEM+10% horse serum (Sigma, lot #90H-701 Sigma, St. Louis, Mo.) withpenicillin-streptomycin antibiotic (Gibco, Long Island, N.Y.). Thesuspension was then filtered a through 20-μm NITEX filter and an aliquotof the cells counted on a hemocytometer. The cells were plated at 105per 100 mm gelatin-coated culture dish (Falcon, Norcross, Ga.). Thesecultures were termed "primary culture".

The cells were maintained for 7-10 days with media changes every 3 daysuntil the cell layer was confluent. The cells were then detached fromthe dish with 0.025% trypsin in a solution of 3:1 Dulbecco'sphosphate-buffered saline (DPBS) without Ca²⁺, Mg²⁺, and DPBS-EDTA. Thetrypsin was neutralized with horse serum and the suspension wascentrifuged at 150×g for 20 minutes. The supernatant was discarded, thecell pellet resuspended in EMEM+10% horse serum and the cells filteredthrough a 20 μm nitex filter. The cells were counted on a hemocytometer,the concentration adjusted to 2×10⁶ cells/ml, and 0.5 ml of cellsuspension placed in a cryovial to which was added 0.5 ml of 15%dimethylsulfoxide (DMSO) in media (final concentration of 7.5% DMSO).The cells were then placed in freezing chamber (Fisher) and slowlyfrozen to -80° C. After at least 16 hours, the cells were thawed andplated at 100,000 cells per 100 mm gelatin-coated culture dish and grownto confluence. This is termed "secondary culture" and consists ofmesenchymal stem cells.

The cells were released from the dishes with trypsin treatment andcultured on polymer scaffolds in 35 mm tissue-culture treatedpolystyrene dishes. Each disk was initially seeded with 4×10⁶ cells in avolume of 100 μl. Samples were incubated at 37° C. in a humidifiedatmosphere containing 5% CO₂ to permit cell adhesion to, and entrapmentwithin, the polymer scaffold; 2.5 ml of culture medium were thencarefully added after 6 hr, and 1.5 ml after 24 more hr. Medium wasreplaced every 2-3 days for 21/2 weeks of tissue culture.

The full thickness cartilage defect was made according to the proceduredescribed by Freed and Grande, J. Biomed. Mater. Res. 28, 891 (1994).New Zealand white rabbits were used according to N.I.H. guidelines forthe care of laboratory animals (N.I.H. publication #85-23 Rev. 1985).The rabbits, 8 month old males weighing approximately 4.5 kg, wereplaced under general anesthesia with xylazine (5 mg/kg i.m.), andketamine (35 mg/kg i.m.), then shaved and scrubbed with betadine. Amedial parapatellar arthrotomy was performed bilaterally with the rabbitsupine. A pointed 3 mm diameter custom drill bit (Acufex, Mansfield,Mass.) was used to create a full thickness defect (1-2 mm deep) in thefemoropatellar groove (FPG). An attempt was made to extend this defectjust through the subchondral plate without violating the subchondralbone. A surgical trephine (Biomedical Research Instruments, Wakerville,Md.) was used to core a 4 mm diameter×2 mm thick piece of PGA matrix,and this was press-fit into the 3 mm diameter defect in the rabbit'sFPG. The incision was closed in two layers; the fascia was closed withinterrupted 4.0 VICRYL® (absorbable) and the skin was closed with thesame interrupted 4.0 VICRYL®.

The knee joints were not immobilized postoperatively, and the animalswere allowed free cage activity. Rabbits were euthanized after 6 and 12weeks using an overdose of pentobarbital. The protocol for euthanasiawas 1 cc of Ketamine intramuscularly (i.m.), wait fifteen minutes, then5 cc of SLEEP-AWAY® i.m. (Fort Dodge Lab, Fort Dodge, Iowa).

Four adult male rabbits had PGA-stem cell implants into their right kneewhile the left knee served as a control with only the PGA matrix beingimplanted. Two rabbits were euthanized at six weeks and other tworabbits euthanized at twelve weeks.

Joint repair was assessed histologically as follows. Knee joints wereharvested, fixed in formalin, and decalcified in 5% nitric acid for 5-7days, with daily changes of the nitric acid, and bisected in a coronalplane through the center of the defect. Samples were then processed forembedding in paraffin, sectioned (6 μm thick), and stained usinghematoxylin & eosin or Safranin-0, then viewed at 25 to 200×magnification by light and phase contrast microscopy.

Results

Mesenchymal stem cells were isolated from rabbit muscle and grown toconfluence in culture. These cells have the characteristic mononuclear,stellate shape associated with MSCs isolated previously from chick andrat skeletal muscle. When the MSCs were cultured in the PGA matrix, thecells adhered to the matrix but did not differentiate. There was noapparent cartilage matrix.

The defects containing PGA polymer alone (control) at six weeks show asmall amount of matrix from the dissolving PGA disc among the abundantfibrous elements. There was a definite boundary between the implant andthe host cartilage with no presence of articular cartilage in theimplant. There were non-specific fibrocartilage cells and abundantcollagenous matrix in the control at 6 weeks. The PGA-MSC matrices atsix weeks resembled the controls. There were relatively undifferentiatedcells and nests of apparent cartilage. Remnants of the PGA matrix werealso present.

Normal articular cartilage was present adjacent to the defect. The cellscould be seen in lacunae. The proliferative zone with isogenous nestswas clearly visible, as is the tidemark. At twelve weeks, the controlshad a patchy mixture of fibrous and hyaline cartilage and extensiveconnective tissue. There was no evidence of new subchondral bone. Thesurface layer of cells appeared fibroblastic, lacking the round lacunaecharacteristic of hyaline cartilage, and were oriented perpendicular tothe surface.

In contrast, the PGA-MSC matrices at 12 weeks showed a surface layer ofcartilage approximately the same thickness as the host cartilage andnormal appearing subchondral bone. The surface layer of cartilagecontained chondrocytes within lacunae surrounded by cartilaginousmatrix. Isogenous nests could be discerned. There were also islands ofapparent mesenchymal stem cells. Beneath the cartilage, a tidemark wasseen in places. Trabecular, cortical bone underlay the cartilage,complete with hematopoietic tissue. There was good integration of thetissue in the defect with the surrounding tissue.

Discussion

The data shows the successful regeneration of articular cartilagedefects using mesenchymal stem cells harvested from rabbit muscle. TheMSCs were obtained from rabbit muscle. When cultured in media, the MSCsmaintain an undifferentiated phenotype but, when treated withdexamethasone, they differentiate into a number of mesodermalphenotypes. This behavior in vitro is identical to MSCs isolated fromrat and chick.

There is little difference between control and experimental defects at 6weeks post-op. However, by 12 weeks post-op, there are dramaticdifferences between the two treatments. The PGA-MSC matrix at hassimilar histology to normal cartilage. A layer of cartilage andsubchondral bone are evident and it is difficult to ascertain the edgeof the experimental implant even at 200× magnification. The experimentaldefects had a good, but not perfect, articular surface, with occasionaldefects at higher magnifications. The surface is the same thickness asthe surrounding cartilage. The subchondral bone, however, isindistinguishable from that seen in normal articular cartilage, and itis impossible to determine the base of the defect.

In contrast, the control defects at 12 weeks post-op are filled withapparent fibrocartilage. This is particularly evident in the areaadjacent to the surface. Deeper, the defect contains chondrocytes inlarge lacunae. There is no evidence of subchondral bone, and theinterface between defect and surrounding tissue is obvious.

It appears that the MSCs differentiate into chondrocytes and osteoblastswithin the defect. It appears the MSCs differentiate in such a manner asto re-create the spatial orientation of the tissue, cartilage at thesurface and bone underneath. The signals mediating this differentiationare unknown. Presumably, the cells respond to endogenous signalsemanating from the surrounding cartilage and bone, although othersources such as synovial fluid and blood cannot be eliminated. There mayalso be mechanical signals, although the defect site in thefemoropatellar groove is not weight-bearing.

The present study indicates that MSCs are useful in the regeneration offull-thickness articular cartilage defects.

Modifications and variations of the present invention will be obvious tothose skilled in the art from the foregoing description. Suchmodifications are intended to come within the scope of appended claims.

We claim:
 1. A method for growing new articular cartilage or articularcartilage and subchondral bone in a patient comprisingadministering to asite where articular cartilage or articular cartilage and subchondralbone is needed isolated mesenchymal stem cells seeded in a polymericcarrier suitable for proliferation and differentiation of the cells intoarticular cartilage or articular cartilage and subchondral bone, whereinsaid isolated mesenchymal stem cells are purified pluripotentmesenchymal stem cells, which cells are characterized by beingsubstantially free of multinucleated myogenic lineage-committed cells,and which cells are predominantly stellate-shaped cells, wherein themesenchymal stem cells form predominantly fibroblastic cells whencontacted with muscle morphogenic protein in tissue culture mediumcontaining 10% fetal calf serum and form predominantly branchedmultinucleated structures that spontaneously contract when contactedwith muscle morphogenic protein and scar inhibitory factor in tissueculture with medium containing 10% fetal calf serum.
 2. The method ofclaim 1 wherein the mesenchymal stem cells are isolated from muscle ordermis.
 3. The method of claim 1 wherein the polymeric carrier isbiodegradable.
 4. The method of claim 1 wherein the polymeric carrier isformed of polymer fibers as a mesh or sponge.
 5. The method of claim 4wherein the polymeric carrier is a polyglycolic acid fibrous mesh. 6.The method of claim 4 wherein the polymer is selected from the groupconsisting of proteins, polysaccharides, polyhydroxy acids,polyorthoesters, polyanhydrides, polyphosphazenes, and combinationsthereof.
 7. The method of claim 1 wherein the polymeric carrier is ahydrogel formed by crosslinking of a polymer suspension having the cellsdispersed therein.
 8. The method of claim 7 wherein the polymericcarrier is selected from the group consisting of polysaccharides andsynthetic polymers.
 9. The method of claim 1 wherein the defect is inboth cartilage and bone.
 10. The method of claim 1 wherein themesenchymal stem cells in the polymeric carrier are implanted in anosseous site.
 11. The method of claim 1 for growing new dense fibrousconnective tissue comprising implanting the mesenchymal cells in apolymeric carrier at a site in need thereof associated withreconstruction of the spine, pubic symphysis or temporomandibular joint.12. A method according to claim 1 for growing new mixed connectivetissue comprising implanting said mesenchymal cells in a polymericcarrier in a fascial plane formed of muscle cells, fat, fibroblasts, andcartilage.
 13. The method of claim 1 wherein the mesenchymal stem cellsare seeded onto and into the biodegradable polymer carrier sixteen hoursto two weeks prior to administration to the site.
 14. The method ofclaim 1 wherein the cell density of the mesenchymal stem cells at thetime of implantation is approximately 1 to 20 million cells per ml. 15.The method according to claim 1, wherein the new articular cartilage orarticular cartilage and subchondral bone is formed in the knee joint.